Højrup Peter
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark.
Methods Mol Biol. 2015;1348:65-76. doi: 10.1007/978-1-4939-2999-3_8.
Amino acid analysis is a highly accurate method for characterization of the composition of synthetic peptides. Together with mass spectrometry, it gives a reliable control of peptide quality and quantity before conjugation and immunization. Peptides are hydrolyzed, preferably in gas phase, with 6 M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by ion-exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and cysteine also, unless derivatized, and the amides, glutamine and asparagine, are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.
氨基酸分析是一种用于表征合成肽组成的高度准确的方法。与质谱法一起，它能在偶联和免疫之前对肽的质量和数量进行可靠的控制。肽被水解，最好在气相中，用6 M盐酸在110°C下处理20 - 24小时，然后通过柱后茚三酮衍生化的离子交换色谱法分析所得氨基酸。根据水解条件，色氨酸会被破坏，半胱氨酸也是如此，除非进行衍生化，并且酰胺、谷氨酰胺和天冬酰胺会分别脱酰胺化为谷氨酸和天冬氨酸。文中提出了三种不同的计算结果的方法，考虑到上述局限性，通常可以获得优于5%的定量结果。
